Rigid non-swellable polymeric materials having neutral, hydrophilic surfaces are useful for many applications protein solutions. These include chromatography supports, membranes, carriers for immobilized enzymes or immunoassay supports. Hydration of polyacrylonitrile surfaces to form acrylamide groups is well known in the art.
Stoy, U.S. Pat. No. 4,110,529, discloses introducing reactive groups into the surface layer of beads during coagulation. Example 5 of the Stoy patent discloses the partial hydration of a polyacrylonitrile to 40 percent amide groups and then coagulating to form porous beads. However, beads prepared in this manner are highly swellable in water and contain a substantial amount of byproduct carboxylate groups in addition to the desired amide groups. Thus, the beads are not particularly useful as chromatographic supports. Their tendency to swell results in excessive pressure drops and inconsistent flow rates in chromatographic columns and the presence of carboxylate groups causes non-specific binding in separation processes not involving ion exchange. Problems also arise from the high, 40%, amide conversion rate as high conversion to amide groups results in significant losses in chromatographic flow due to loss of bead rigidity.
Other attempts to convert nitrile groups to amides in the prior art have involved treatment with strong acids or bases. Both of these techniques generally lead to some formation of surface carboxyl groups Rigopolous, U.S Pat. No. 4,143,203 discloses solid particles possessing an impermeable rigid polyacrylonitrile core with a hydrolyzed surface. The surface is hydrolyzed by heating the solid poly-acrylonitrile particles in a solution of sulfuric acid at temperatures ranging from 75.degree. to 95.degree. C. However, beads formed under these conditions are non-porous and have a substantial amount of byproduct carboxyl groups; and thus are not useful in non-ion exchange protein specific chromatographic applications.
The surface modification of polyacrylonitrile under basic conditions was studied by K. Ohta et. al., Nippon Kagaku Kaishi, 6, 1200 (1985) using surface infra red spectroscopy. After treating polyacrylonitrile films with 5 percent sodium hydroxide for 4 hours at 70.degree. C., Ohta found 4.5 percent amide and 5.7 percent carboxylate groups on the surface. Treatment of the film with 5 percent sodium hydroxide and 15 percent hydrogen peroxide (an aqueous alkaline peroxide reaction) for 4 hours at 70.degree. C. gave 2.1 percent amide and 0.7 percent carboxylate. These treatments are also not sufficiently selective.
Thus, the present state of the art still possesses serious drawbacks to the formation of highly selective non-swellable highly porous acrylonitrile polymeric beads having neutral hydrophilic surfaces. The greater surface area of highly porous beads and the narrow diameter of the polymer structure, makes it critical to accurately control the extent of hydration. Conversion of more than 10 percent of the nitrile groups to amide groups results in significant losses in flow in chromatography separations. It is difficult to accurately control the extent of reaction with acidic hydration. Acidic hydration is also known to have a strong neighboring group effect which generates a "block" polymer structure. A block polymer structure at low conversion can result in non-uniform coverage of the surface. Again, this causes problems with non-specific binding in chromatography applications. A third problem with acidic hydration is the formation of carboxyl and imide groups. The presence of carboxyl groups causes undesired ion interactions during size exclusion or affinity chromatography applications.
Surprisingly, the present inventors have found that alkaline peroxide hydration of nitriles, with careful control of the solvent can avoid these problems. The reaction selectively converts nitriles groups to amide groups without side reactions to imide or carboxyl groups. By controlling the solvent, the reaction can be easily controlled and actually stopped at low conversion. The use of solvent, preferably methanol, allows all of the surfaces of the polymer, even those present in small pores, to be converted. This results in an even distribution of amide groups on the surface of the polyacrylonitrile core. Also discovered is a method by which the amide groups, the nitrile groups, or a combination thereof can be reduced to generate amine functionality. Furthermore, a method has been found to succinylate the amine groups to generate carboxyl functionality. The attachment of a bioactive ligand by carbodimide activation of the carboxyl groups has been found to effectively form complexes with enzymes, hormones, or mixtures thereof for biological separation techniques.
Furthermore, the rigid nature of the polyacrylonitrile core is minimally effected by this mild treatment and thus, the products of this invention are substantially non-swellable in water and noncompressible. When used herein and in the appended claims the term "non-compressible" means able to resist hydrostatic pressures in columnar beds of up to about 3000 psi, without collapsing.